JP2007515958A - Cultured cells, cell culture methods and equipment - Google Patents

Abstract

  Cell culture can be performed by constraining one or more cells by a barrier. The distance between the barriers is approximately equal to the size of the cell to be cultured. The space between the barriers is narrow enough to control or monitor the properties of the cells cultured there. Cells can be fully constrained or move between two opposing barrier surfaces. The gap between the two opposing barriers is sufficiently narrow that only the cell monolayer is cultured. The barrier may be transparent. The surface of the barrier may have one or more preselected characteristics that mimic the characteristics of the biological niche of the cell. The number of cells during cell culture is limited, and the characteristics of individual cells can be controlled. Cultured cells may be monitored over time, such as by imaging using standard bright field or fluorescent imaging techniques.

Description

  The present invention relates generally to cell culture, and more particularly to cultured cells, cell culture methods and equipment.

  Normally, cells are cultured in vitro by growing in a fluid or gel-like growth medium in a sterile container under controlled conditions including temperature and pH. In many stem cell cultures, for example, cells grow as free-floating aggregates in a suspension of media. However, in such culture, the microenvironment in the immediate vicinity of the cells changes constantly, and the cells are exposed to various uncontrolled environments, and the cellular in vivo environment is not much simulated. As a result, even when culture is started with a single cell, a heterogeneous mixture of cell types occurs.

  Molecules secreted by cells in vivo are important for the function of those cells and the function of neighboring cells. However, in conventional culture systems, these molecules diffuse and dilute rapidly, so the interaction between the cells that occur in vivo and the locally secreted molecules is lost or ineffective, The normal function of one or more cells is affected. Furthermore, in certain normal cell cultures, the cells tend to adhere or adsorb to the surface of the cell culture vessel, which is undesirable for certain cell types. For example, the attachment of many stem or progenitor cells is not preferred because it promotes differentiation or changes the cell phenotype.

  Traditional in vitro cell culture methods and instruments control the characteristics and phenotype of individual cells, track cell division and cell lineage, and monitor the activity (behavior) history of each cell in the culture. It is difficult or impossible to record. Conventional techniques, such as laser scanning confocal microscopy, that produce high resolution images of cultured cells are not suitable for long-term imaging of cultured cells. For example, in conventional high resolution imaging techniques such as laser scanning confocal microscopy, cultured cells are typically exposed to high intensity light. As a result, the time to image live cells is limited due to light or laser-induced phototoxinosis. Moreover, it is usually expensive to obtain a high-resolution image of a culture cell using conventional techniques.

  Thus, there is a need for improved control of cell culture. An object of the present invention is to provide a cell culture method, a cultured cell, and a cell culture device.

  One or more cells are cultured in a space constrained by a barrier. Cells are either fully constrained or move horizontally between two opposing barrier surfaces. The gap between the two opposing barriers is sufficiently narrow that only the cell monolayer is cultured.

  It is clear that culturing cells in a constrained space is significant. For example, the environment of each cell can be accurately controlled, and a culture environment reflecting a biological niche can be provided. It is also possible to remove special cells from the aggregate of cultured cells or add cells to the aggregate of cultured cells. In addition, because cells are constrained between the barriers, they can live without imaging using confocal microscopy or laser imaging equipment, using imaging techniques such as standard brightfield imaging or fluorescence imaging. It becomes easier to observe cell growth directly in detail with sub-cell dimensions. When the cells are constrained in the vicinity of the focal plane of the objective lens, the image quality is significantly improved compared to the image of the cells away from the focal plane.

  Accordingly, in an aspect of the present invention, a cell culture method is provided. In this method, the cells are restrained between the first and second barriers. The barriers are separated by a distance about the size of the cell so that the cells come into contact, and the cells are prevented from moving toward or away from each of the first and second barriers. The

  In another aspect of the invention, a method for controlling the properties of individual cells in a cell culture is provided. In this method, cells are constrained in a space defined by at least two surfaces that contact the cells. This space has dimensions similar to cells. Each surface that the cell contacts has one or more preselected properties. The number of cells surrounding the cell is limited to such an extent that the characteristics of the cell can be controlled. Culture material is installed in the space. The preselected characteristics may be selected to mimic the characteristics of a cell's biological niche.

  In any of the foregoing methods, a plurality of cells may be cultured between the barrier or surface. A plurality of cells may form a monomolecular layer. The cultured cells may be monitored using, for example, an optical device or a sensor. The result of the monitor may be recorded. Alternatively, additional cells adjacent to the cultured cells may be manually placed so that the two cells interact with each other.

  In another aspect of the invention, a method for forming an artificial tissue is provided. The method comprises the steps of placing a cell monolayer on another cell monolayer, and allowing cells in one monolayer to interact with cells of another monolayer. . The monolayer of each cell is cultured by the method of the present invention.

  In another aspect of the invention, there are provided cells cultured by the methods of the invention or cell aggregates having such cells. The cell or cell aggregate may be used for artificial tissue, organ, cell transplantation or in vitro fertilization.

  In another aspect of the invention, a combination cell culture device and cell culture is provided. This combination has a container that defines a chamber chamber that contains a fluid culture medium, and at least two partition walls that define a space in the chamber chamber. Cells are housed in the space in continuous contact with each of the barriers during culture. Each barrier has one or more preselected properties. This combination has means for providing culture material in space at a predetermined rate, the means having one or more fluid passages that allow fluid to flow into and out of the chamber chamber. . Means for adjusting the flow rate of the fluid flowing in or out of the chamber chamber may be provided. In addition, a means for monitoring cells housed in the space may be provided.

  In another aspect of the invention, a combination cell culture device and cell culture is provided. The combination includes a container that defines a chamber chamber that contains the fluid culture material and at least two barriers that define a space within the chamber chamber. Each barrier has one or more preselected properties. Two or more cell aggregates are housed in the space such that the aggregates are maintained in continuous contact with each of the barriers during culture. This combination also has means for providing the culture material in space at a predetermined rate. The aggregate may have one or more cell layers. For example, the assembly may have a monolayer of cells.

  In another aspect of the invention, an apparatus for culturing cells in a controlled artificial space is provided. The apparatus has a container that defines a chamber chamber that contains a fluid culture medium, and at least two barriers that define a space in the chamber chamber for cell culture. The inner surface of each barrier has one or more preselected properties. The space is small enough to control the characteristics of one or more individual cells cultured in the space. The apparatus also has means for providing culture material to the space.

  In another aspect of the invention, a plurality of devices are provided. Each device is one of the above-described devices and combinations, and is combined with each other to form a cell culture assembly. Each of the plurality of devices has a substantially plate shape, and the devices are stacked in parallel.

  In another aspect of the invention, a method of culturing cells is provided, the method comprising culturing one or more cells while restricting cell migration, each cell having two opposing Continuous contact with the barrier surface. However, cells can move between barrier surfaces. During culture, the cells are imaged using a non-confocal imaging device, such as a bright field imaging device or a fluorescence imaging device, including a differential interference contrast (DIC) imaging device.

  Other aspects, features and advantages of the present invention will become apparent to those of ordinary skill in the art by reference to the following description of specific embodiments of the invention with reference to the accompanying drawings.

  The figure is shown to illustrate one embodiment of the present invention.

  The inventors of the present application have found that in vitro cell culture is significantly controlled by controlling the microenvironment of individual cells in the culture chamber. The microenvironment of a cell is a local environment that surrounds the cell or the immediate vicinity of the cell, also known as a niche. In the present invention, the biological environment in a living body is called a “natural niche” or synonymously “biological niche”, and the in vitro microenvironment in cell culture is called an “artificial niche”.

  The inventors have identified certain aspects of an artificial niche of cells. This aspect needs to be controlled by an artificial niche to simulate a biological niche. One such embodiment is the size and shape of an artificial niche that is at least partially shaped by an adjoining cell contact; another embodiment is the physical and surface of the surface forming the artificial niche that surrounds the cell. Another aspect is the supply of nutrients to the artificial niche and the removal of metabolites from the artificial niche. In addition, monitoring and recording the characteristics of each individual cell and its microenvironment facilitates the cultivation of individual cells having the desired characteristics, such as the desired phenotype.

  In an organism, the cellular microenvironment is unique to each cell type and is almost unique to each cell; thus, it is possible to obtain complex patterns, such as the human body. The inventors have identified the requirements that the microenvironment of a cell must meet in order to control cellular properties such as phenotype and cell differentiation. In cell culture, when these requirements are satisfied, control with higher accuracy than conventional systems becomes possible. In some cases, cultured cells can be more accurately controlled in an artificial niche than biological niches. For precise control of cell culture, the microenvironment must meet the following requirements in a manner appropriate for the particular cell type: 1) The niche has the appropriate dimensions and shape; 2 ) The solid, liquid, gas or other phase that forms the niche boundary and interior has the appropriate chemical and physical properties; 3) The niche has the appropriate solution source (source) and reservoir (tank) Have. Artificial niches and individual cell characteristics are preferably monitored. The inventors have created an artificial niche that meets these requirements.

  In culture, artificial niche exists alone or as an adjacent group. In other words, the culture device may have two or more population niches. Unlike the mass of cells where the microenvironment surrounding each cell during bulk culture is unregulated and uncontrolled, the artificial niche formed in the embodiments of the present invention is shaped and controlled. By shaping and controlling the boundaries surrounding each cell, a large group of cells can be obtained and the solution can diffuse into the group so that the niche surrounding each cell in the group is effectively shaped and controlled. become. Monitoring removes unwanted cells (eg, by photoablation) and regenerates the function of the organism's immune system.

  Separate niches or adjacent groups of niches may also be connected to each other by grooves, which allows some cells in the culture device to be chemically protected while not in physical contact with other cells. Connection becomes possible. The culture device may have any combination of separate niches, niche assemblies, and niches that are physically contactless, chemically or otherwise connected.

  In particular, the inventors have encapsulated or confined cells within a predetermined space, such as between two or more extracellular barriers or between adjacent cells, where the space is a cell or its biological niche. By having the same dimensions as the dimensions of the artificial niche, the characteristics of the artificial niche can be effectively controlled and / or monitored. When cultured cells are contained in a predetermined space, the artificial niche can be controlled to be close to the model of the biological niche. For example, the constraining barrier may be shaped to the desired size and shape of the space, may have the desired surface characteristics, and may be connected to a suitable source and supply tank. In addition, when cells are constrained, cell movement is restricted, so that the growth and division of individual cells can be monitored. In many embodiments, it is preferred that the cells of interest be constrained or limited on all sides, but in certain embodiments, the pre-shaped space contains cells or their biology in order to obtain the advantages described above. Obviously, it is only required to have dimensions comparable to the dimensions of the target niche.

  The cell is constrained between two barriers that are separated by a distance equal to the cell's dimensions, and the barrier touches the cell and inhibits the cell from moving toward and away from each barrier. Is done.

  The barrier is thus formed by at least two separate surfaces in contact with the cells, which is distinguished from a single continuous barrier that forms the perimeter of the cells, for example by implanting the cells in a continuous gel.

  The barrier suppresses cell movement relative to the barrier. The barrier may be composed of any rigid non-cell material and can be in various shapes. The barrier is permeable, selectively permeable, or impermeable. For example, the barrier may be a glass plate, a porous membrane, or a gel. The barrier functions to create a microenvironment where the desired rules, phenotype, behavior, function or proliferation of the cell can be easily obtained, and the selection of an appropriate barrier depends on the specific purpose of the cell culture This will be apparent to those skilled in the art from the following description.

  The minimum distance between the barriers is equivalent to the size of the restrained cell, which suppresses cell movement, while the cell is not over-compressed, causing the cell to be adversely affected or accidentally affected For example, it is compressed to the extent that it does not become damaged, die, or exhibit an abnormal function.

  As shown below, in certain embodiments of the invention, the microenvironment of the cell is at least partially controlled by controlling the physical, chemical and biological properties of the surface in contact with the cell. The microenvironment of the cells can also be at least partially controlled by controlling the method of providing and dispersing the culture material in the cell niche. For example, in the case of a fluid culture medium, the direction and flow rate of the fluid flow, the concentration of certain components in the medium, and the concentration gradient are controlled and adjusted. The culture material includes a culture fluid having growth factors, nutrients, (dissolved) oxygen, and the like. The fluid flow is adjusted, for example, a concentration gradient is generated. It is clear that fluid flow can be controlled by adjusting the diffusion flux or convection velocity or both. It is clear that the culture material can be provided to the cell niche in various ways and in various structures. For example, the fluid is supplied through a fluid channel, a permeable plate or an opening in the culture chamber. The culture material may also be provided to the cells by other cells.

  It is obvious that cultured cells having desired characteristics can be obtained by controlling an artificial niche by the above-described method and sufficiently simulating a biological niche.

  As further shown below, in certain embodiments of the present invention, cell growth and division can be monitored (eg, optically) and recorded based on individual basal cells. The lineage history of each cultured cell can be monitored and recorded. Thus, specific information regarding the characteristics of each individual cell can be obtained. Accurate information on cell properties and history is useful in controlling cell cultures and in future application of cultured cells.

  Specific embodiments of the present invention, including methods and apparatus, are described below with reference to the accompanying drawings.

  Referring to FIG. 1, an exemplary embodiment of the present invention includes a cell culture device 20. The device 20 includes a base plate 22 and a tube 24 attached to the base plate 22, thereby forming a container 25 that defines a chamber chamber 26 that receives and houses the liquid culture medium. Furthermore, the device 20 may have a cover (not shown) removably attached to the hinge of the tube 24, thereby preventing impurities from entering the chamber chamber formed by the container. The In another embodiment, the cover is loosely attached to the transparent glass or transparent membrane to allow gas exchange.

  A cover plate 28 is installed in the chamber chamber 26 above the base plate 22 so as to face the base plate 22. As shown below, the base plate 22 and the cover plate 28 are installed so as to be separated from each other by a desired distance, and a niche chamber chamber 30 in which cells are contained and restrained is formed between them. . As shown below, the base plate 22 and the cover plate 28 constitute a barrier that accommodates one or more cells between them, thereby allowing the cells to move toward or away from the plate. It is not possible to keep it in contact with the board. The barrier surface may be substantially flat or curved.

  With reference to FIGS. 2 and 3, the base plate 22 and cover plate 28 are held in position by an adhesive, such as a set of silicone adhesive gussets 32 at two opposite end positions of the plate 28. Alternatively, the plates may be held together by other chemical or mechanical holding mechanisms. The cover plate 28 is detachably attached by the hinge of the base plate 22. For example, one end of the cover plate 28 is hinged to the base plate 22 and the opposite end is clipped to the base plate 22. Further, the niche chamber 30 may be shaped by a side plate (not shown) combined with the base plate 22 and the cover plate 28 to form a sealed chamber. In this embodiment, the sealed chamber is One or two or more openings are provided, and communication between the niche chamber 30 and the chamber 26 is possible through the openings (when there is no side plate, the base plate 22 and the cover plate 28 are connected to the chamber chamber). It is clear that the open niche chamber 30 communicating with 26 is shaped).

  Plates 22 and 28 should be close enough to avoid unintended consequences such as undesired cell damage, accidental cell differentiation, cell response or changes in cell function. Obviously, it is necessary to restrain the cells between them by slightly compressing the cells so that they do not occur.

  Suitable materials for the construction of the containers that shape the culture chamber will be apparent to those skilled in the art. For example, the tube 24 may be made of glass or plastic such as polystyrene. Transparent materials are significant because they allow for optical monitoring of cells. The tube 24 may be any desired size or shape and may be large enough to accommodate a plurality of niche chambers.

  The plate 22 and the plate 28 may have various sizes, shapes, and thicknesses. The thickness of the base plate 22 is selected so as to satisfy the requirements for cell monitoring, for example, so as to be thinner than the maximum workpiece distance of the imaging apparatus. The thickness of the plate 22 may be the thickness of a normal microscope cover slip. The plate 22 and the plate 28 may be sufficiently thick so that sufficient rigidity is obtained. However, in other embodiments, one or both of plates 22 and 28 may be flexible, as will be described in detail below.

  The plate 22 and the plate 28 may be made of the same material or different materials. Suitable materials are (porous) glass, contact lens materials, glass polystyrene mixtures, polyethylene, natural or synthetic biocompatible polymers, or other biocompatible materials selected based on the cells to be cultured, these Is shown below. For example, the plate 22 and the plate 28 may be made of untreated bioinert glass or untreated polystyrene.

  Before or during culture, one or more plates 22 and 28 may be coated or coated with extracellular matrix molecules, adhesion ligands, growth factors, receptors, and the like. It will be apparent to those skilled in the art that the use of a coating on the inner surface of the culture chamber is significant.

  It is clear that cells can adhere to the surface they come into contact with, whether preferred or not. In order to suppress cell adhesion, the inner surface of the plate is made of an adsorption-suppressing substance or coated with an adsorption-suppressing substance. Examples of adsorption-inhibiting substances include untreated polystyrene, glass, polyacrylamide, gels, or anti-adsorbed biomolecules including polysaccharides, proteoglycans, proteins, or polyethylene oxide. Thus, the cells can be restrained without necessarily adhering or adsorbing to the plates 22 and 28. For example, one or both of plates 22 and 28 may be coated with a material that affects cell adsorption and behavior, such as poly (dimethylacrylamide) or dimethyldichlorosilane.

  It is also possible to control cell growth using an inner surface that has an affinity for certain types of cells. When the surface has no or little affinity for the cultured cells, the cultured cells are not adsorbed on the surface of the cover plate even if they are restrained. For this reason, the cells become mobile and can move horizontally along the inner surface of the plate. For example, the cells may be moved by a concentration gradient in the culture medium, or the cells may be moved by an external force such as a magnetic field. The cells may be oriented in a certain direction depending on the surface properties. Surface materials may also be selected to evaluate compounds that attract or excrete certain cells.

  The inner surfaces of plates 22 and 28 (meaning the inner surface in contact with the cells) may in particular be composed of materials selected to promote or inhibit the growth of certain cells.

  Each plate 22 and 28 (and side plate, if present) may be gas permeable (or impermeable). The gas permeable plate is required when it is preferable to allow gas to permeate the niche chamber 30. The cover plate 28 may also be permeable to a given component of the liquid culture medium so that the component is transported through the plate 28 to the niche chamber 30 or alternatively or in addition thereto. In addition, the niche chamber 30 may be provided with an opening. Plates 22 and 28 may be transparent or opaque. The transparent plate facilitates optical monitoring of cells in the culture and allows the promotion of certain chemical or biological changes using light in the culture chamber or cells.

  The plates 22 and 28 may be very hard, soft and flexible depending on the cells to be cultured and the intended application. A hard barrier can be used when the cells are restrained accurately. Soft barriers can be used in applications where it is desirable to stretch or grow cells within a limited range of dimensions, such as cultured bone cells, lens cells and other hard tissues. The cover plate may be made of a shape memory material or a photoelectric shape sensitive polymer. Such a cover plate is used when it is preferable to change the distance between the barriers during cell culture. For example, the cover plate is used when the compressive force or tensile force applied to the cells changes periodically.

  The plates 22 and 28 may have various properties in a given application, as described above, but both plates in the given application have an overall microenvironment of cells confined within the niche chamber 30. In order to improve the control, it can also be selected to have certain features.

  The distance between the two plates is equivalent to the size of a single cell confined in the niche chamber 30 and the cells grow as a monolayer in the niche chamber. In that case, the distance varies depending on the size of the cell to be cultured. In another embodiment, the distance between plates 22 and 28 is about 0.05 to 250 microns, of which the lower range is more suitable for bacterial cultures. For example, when culturing stem cells, this distance is in the range of about 2 to 40 microns.

  As will be apparent to those skilled in the art, growing cells within a monolayer is significant. For example, it makes it easier to monitor cells in culture for the following reasons; shortens the light path across the monolayer; controls the niche of the target cell (eg, installs or removes adjacent stromal cells) Direct manipulation of adjacent cells; accurate positioning of source and reservoir relative to target cells.

  If the gap between plates 22 and 28 is dimensioned to limit monolayer cell growth, a large monolayer such as a growth factor or cytocan introduced into the culture chamber 26 is Concentration gradients between the bulk medium and the cells placed away from the opening between the plates, conveniently diffuse only into the niche chamber 30 at a controlled rate compared to small molecules such as oxygen and glucose Occurs. In vivo, concentration gradients occur, so it is preferable to create such gradients when mimicking a biological microenvironment in certain applications. If the plates 22 and 28 are separated by a distance equivalent to the dimensions of a single cell, the gradient that occurs for some large molecules is preferably approximately the gradient of these molecules in the biological niche. Correspond.

  The number and size of the openings in the niche chamber 30 can be dynamically adjusted to meet the specific requirements of a specific application, if necessary. For example, the niche chamber 30 may be sealed or partially sealed, for example, by providing one or more side plates (not shown). The side plate has one or more openings. The side plate may be permeable, impermeable or selectively permeable. The openings in the side plates may be of various dimensions from nanometer-scale pores to cell-scale grooves that allow cells to move or stretch from the niche chamber 30.

  When the side plate is not present or when the side plate has the same opening, a symmetric concentration gradient is formed in the niche chamber 30. When only one side plate is included, or when an asymmetric opening is provided in the side plate, a relatively long concentration gradient of macromolecules is formed in the niche chamber 30 so that in vivo The concentration gradient experienced by a particular cell type is reformed. Further, the container 25 has a weir adjacent to the opening of the niche chamber 30 and the bulk medium is partitioned, and the niche chamber 30 has a large unidirectional concentration gradient. The strength and time of the gradient both depend on the inherent cell consumption rate, the diffusion molecular size, the molecular concentration in the chamber 26 and the distance between the barriers.

  The plates 22 and 28 are substantially parallel or slightly inclined. An inclined niche chamber is preferred when forming a pie-like niche and when promoting cell movement along a narrow axis. Having a wide range of dimensions with a continuous gradient within a single field of view of the microscope is beneficial when assessing the effects of a range of cell gap dimensions. For example, an angle in the range of 0 to 45 degrees is used.

  The device 20 may be composed of readily available materials. A standard microscope cover slip may be used as the base plate 22, and a small piece of microscope slide may be used as the cover plate 28. They use biocompatible elastic adhesives such as silicone adhesives or dental adhesives, along at least two edges of the cover plate 28, or some combination of positions along the outer periphery of the plate 28 And bonded to each other. To form a niche chamber 30 of the desired size or volume, the cover plate 28 is separated from the base plate 22 by spacers during installation of the adhesive, or if elastic glue is used, the cover plate 28 is It is separated from the base plate 22 after the adhesive is installed. Tubes of any size or shape may be constructed using standard materials for tissue culture such as plastic, glass or silicone, and this tube is glued to the top of the base plate 22 to form the container 25. The

  If necessary, one or more fluid conducting tubes (not shown) may be connected to each position of the chamber chamber 26 to allow fluid to flow through and from the chamber chamber 26. The tube extends toward or into the opening of the niche chamber 30. Each tube may have a different dimension and may be controlled separately. Thus, a particular fluid component is transported to a particular location in chamber chamber 26 or niche chamber 30 at a particular desired speed.

  If necessary, the chamber 26 may be divided into two parts having a weir extending over the upper part of the plate 28. The media contained in the two compartments of the chamber 26 are at different pressures or have different component concentrations, and a flow through the niche chamber 30 is formed. This flow is controlled or regulated by controlling the pressure differential or concentration.

  With reference to FIGS. 1, 4 and 5, in operation, one or more cells 36 are seeded and cultured in niche chamber 30.

  Prior to placing cells 36, chamber chamber 26 and niche chamber 30 are sterilized, for example, using an autoclave or by UV exposure.

  If necessary, one or more hard spacers 34 of equivalent dimensions are installed in the niche chamber 30 between the plates 22 and 28 before, during or after the cell 36 is installed. Is done. Suitable spacers 34 include polymers such as polystyrene or glass microspheres. The spacer may be a phosphor. They can also be coated with biomolecules or reactive chemical functional groups. These may be magnetic materials. The spacer 34 has sufficient rigidity and is resistant to compression. The spacer 34 facilitates alignment of the plates 22 and 28 and prevents the cells in the niche chamber 30 from being overly compressed so as to be damaged.

  As indicated above, the plates 22 and 28 preferably do not over compress the cells 36. The inventors found that even if both plates were initially aligned correctly, if the preventive measures were not taken, disturbances during cell culture would cause the plates to be slightly deformed, thereby causing cells 36 between plates 22 and 28 to be Found to be damaged. From this point, it is particularly preferable to install a hard spacer 34 between both plates. By using microspheres as the spacers 34, it is possible to appropriately and accurately install both plates and prevent cells from being excessively compressed. The dimensions of the microspheres can be accurately controlled. The microspheres can be easily dispersed, repositioned or replaced as desired. The microspheres may be composed of any biocompatible material that is sufficiently rigid and that can prevent movement of the plate 28. As described above, in some embodiments, the microsphere is composed of a polymer. In another embodiment, the spacer 32 is formed by integrating one or more plates by embossing or the like on the inner surface of the plates 22 or 28, or the surfaces of both plates.

The spacers 34 and / or cells 36 are installed by various suitable methods. For example, spacer 34 and cell 36 are
By means of injection or capillary action through one or more openings in the niche chamber 30 or by means of a tube inserted into the niche chamber 30, by means of the fluid flowing into the chamber 30. Or separately. These can be directly formed on the base plate 22 after the cover plate 28 is removed or opened. Cover plate 28 can be slightly opened by separating plates 22 and 28 using tweezers and / or temporary spacers. Cells 36 can also be placed using a syringe or pipette. The pipette used has a narrow tip, for example, a cross-sectional area of several microns, and individual cells can be placed with high accuracy. For this reason, the wall of the container 25 has a diaphragm through which a syringe or pipette can be passed through the niche chamber 30.

  If the cover plate 28 is removed or opened during the installation of the cells, the cover plate 28 is carefully re-installed or closed so that the cells are not damaged after installation.

  The medium chamber 26 is filled with a suitable culture medium. The chamber chamber 26 is filled before or after the cells 36 are installed.

  Cells 36 grow and divide in niche chamber 30 under suitable conditions as shown herein or known to those skilled in the art.

  It is clear that plates 22 and 28 compress cells 36 slightly. Accordingly, the cells 36 are physically restrained between the two and are held in the niche chamber 30 and are kept in continuous contact with the plates 22 and 28. Cells 36 cannot move toward or away from both plates.

  Thus, the cells that grow in the niche chamber 30 are physically constrained to a predetermined space by the base plate 22 and the cover plate 28. The gap between the plates is approximately equal to the size of a single cell, and only a monolayer of cells can grow in the niche chamber 30 as shown in FIGS.

  Obviously, because the cells 36 are constrained, it becomes easier to control the characteristics of the cell niche. For example, cells adjacent to the target cell can be selectively placed. The adjacent cells may be one or more specific cell types different from the cell type of the target cell. The concentration gradient of a certain component of the culture medium surrounding the target cell can be accurately controlled. The cells of interest can be continuously monitored and their growth or division history can be tracked and recorded. Plates 22 and 28 also protect cells 36 from the bulk environment and prevent cells 36 from coming into contact with another object or another surface at the top or bottom. The surface properties of the plates 22 and 28 can be preselected, thus preventing the cells 36 from being exposed to surfaces that have uncontrolled or undesirable properties. For example, if the surface of the plate is selected to have properties that mimic the environment to which the cells are exposed in a biological niche, the cells 36 are constantly exposed to surfaces having such properties. Therefore, the controllability of the microenvironment of each cell 36 is improved. Obviously, the preselected characteristics of the barrier may be those inherent in the barrier or may result from a specific treatment of the barrier.

  As described above, when a cell monolayer is cultured, it is possible to remove specific cells from the cell monolayer without causing much disturbance to adjacent cells. It will be apparent to those skilled in the art that removal of specific cells is preferably performed when specific cells are observed that meet, for example, one or more criteria regarding karyotype, morphology and cell size. It is.

  It is clear that one or more cells 36 are cultured with limited cell movement, with each cell 36 in constant contact with two opposing barrier surfaces. However, each cell or whole cell can move between barrier surfaces. That is, the cell can move in a direction substantially parallel to the barrier surface. Preferably, the cells may be imaged during culture using a non-confocal imaging device including a differential interference contrast (DIC) imaging device, such as a bright field imaging device or a fluorescence imaging device. it can. Since the cells are constrained in the vicinity of the focal plane of the non-confocal imaging apparatus, a high-resolution image can be obtained without using a confocal microscope. Thus, the cells 36 can be imaged and monitored over a long period of time (in many days) with little adverse effect on the cells.

  The gap between plates 22 and 28 can be expanded to allow multiple cell layers to grow between the plates, as long as control of the entire individual niche and even individual cell characteristics is possible. good. In most examples, the number of cell layers usually does not exceed 4 or 5. For example, the gap is approximately equal to the total length of two cells. Also, as described above, the inner surfaces of plates 22 and 28 may have different affinities for different cell types. In such a case, the two cell layers are cultured between plates 22 and 28, such that the first layer has the first cell type and the second layer has the second cell layer. Obviously, it will be possible. The two layers of cells usually show similar interactions as in the natural environment.

  The number of cells 36 in the culture chamber 30 or the number of cells surrounding a particular cell 36 is limited by the use and placement of the plates 22 and 28. Limiting the number of cells serves a variety of purposes, such as allowing control of individual cell characteristics or providing improved monitoring of cultured cells.

  Figures 6 and 7 schematically show another embodiment. The cell culture device 40 includes a base plate 42, a cover plate 44, and a large number of side walls 46 that form a niche chamber 48 mutually.

  The base and cover plates 42 and 44 are placed opposite each other at an appropriate distance. This distance is equivalent to the size of the cell to be cultured. The inner surfaces of the plates 42 and 44 are substantially flat or curved. If the dimensions of many cells to be cultured are different, the distance between the plates 42 and 44 will be approximately equal to the maximum cell dimension, or the dimensions will change to accommodate different sized cells at different locations. For example, when sperm cells are cultured, 8 microns is suitable as the minimum distance.

  The side wall 46 is interposed between the base and cover plates 42 and 44 and is installed so as to simulate a desired in vivo cell boundary. It will be apparent to those skilled in the art that the distance between each set of 46 opposing sidewalls will vary depending on the application. For example, when sperm cells are cultured, the distance between the sidewalls varies from 30 to 80 microns.

  One or more inner surfaces of the chamber 48 are shaped to mimic the shape of the biological niche interface. One or more inner surfaces may be non-adsorbable to the cells to be cultured. One or more inner surfaces may be coated with materials such as extracellular fluid matrix proteins, gels, proteoglycans and growth factors to promote cell adsorption and enhance reactivity with other cells.

  The instrument 40 also has a plurality of fluid grooves for fluid movement into and out of the chamber 48, which includes a supply groove 50, source grooves 52, 54, 56 and 58, and a sink groove 60. And 62. The cells are supplied to the chamber chamber 48 via the supply groove 50 using the carrier fluid. The culture fluid containing the growth medium may be supplied to the chamber 48 via the source grooves 52, 54, 56 and 58. Fluids are coated with nutrients, dissolved gases, formulated compounds, tracking dyes, magnetic particles coated with various molecules such as immune bodies, dissolved molecules including molecules normally secreted by the cultured cells, or specific effector molecules. Or a solid or phase identification matrix. Fluid in the chamber 48 containing materials such as carrier fluid, unconsumed growth media, waste, dead cell deposits, etc. is extracted from the chamber 48 through the sink channels 60 and 62. Obviously, the number of grooves will vary depending on the application and manufacturing conditions. For example, a single groove may be used alternately for both the source and sink grooves.

  The details of the groove configuration will vary depending on the function implemented in the particular application. As an example, a simple description of a possible groove structure is shown below.

  The cross-sectional dimension of the groove varies depending on the required flow rate of the carrier fluid. Thus, these are the desired liquid components, such as nutrients or other media supplied to the cells in the chamber 48 or waste and deposits that are produced in the chamber 48 and need to be discharged. Calculated from the quantity. For example, in the case of sperm cell culture, the source groove is structured to simulate blood supply through a capillary. If the source groove has a cross-sectional diameter of 10 microns, the flow rate will be 1 to 100 microns / second, which is sufficient to supply oxygen, for example to 10 cells. In another example, the tubule fluid is fed and drained using the source and sink channels for sperm cell culture, which flows through the flagella of immature sperm to provide nutrition and feed them. It is possible to grow precisely. In one configuration example, a flow rate of 1 pL / second is required, and this flow rate changes during culture in order to simulate biotransformation. Accordingly, the sink groove and the source groove are preferably 10 microns in diameter. In another example, sink and source grooves are used to provide and drain fluid-containing cells, such as cytocans, to mimic the presence of cells that are not present in niche chamber 48 but are present in the body. The In this case, the flow needs to be intermittent and low speed, for example fL (femtoliter) / second. Therefore, the grooves are circular with a diameter of 2 microns.

  A filter or permeable membrane 63 may be placed at the interface between the groove (eg, groove 62 in FIG. 6) and the chamber 48 so that the flow rate is accurately controlled or adjusted, or selected in the fluid Only the substance can pass through.

  In order to adjust or control the flow rate in the groove, a valve 64 may be installed in the groove. Each groove may have a control valve 64. The valve 64 may be of a different type depending on the particular application. The valve is a simple hydraulic type that compresses the incidental groove and deforms the membrane to block the groove, a heating type that expands the same membrane when the gas is heated, and the volume changes greatly depending on the pH The hydrogel may be of a hydrogel pH control type in which the groove is shielded by the hydrogel (the pH is set via an accessory groove), or any other appropriate method. For example, the design of the valve is incorporated by reference in this application, MA Unger et al., “Monolithic Miniature Processed Valves and Pumps with Multilayered Soft Photographic Transfer,” Science, 288, p113-116, 2000; C. Yu et al., “Flux Control of Analytical Microfluidic Chips Based on Thermoreactive Monolithic Polymers and Without Mechanical Parts”, Analytical Chemistry, 75, p1958-1961, 2003; and Q. Yu et al., “Reactive Biomimetic Hydrogel Valves for Microfluids”, Applied Physics Letters, 78, p2589, 2001;

  As described above, one or more sensors 66 may be installed on the base plate 42 (or the cover plate 44) for monitoring. Various sensors can be used. For example, a biocompatible ion reactive field effect transistor (ISFET) based sensor may be used to measure oxygen concentration and pH; a Clark current biosensor may be used to detect glucose concentration and gradient. Also good. A review of chip-type biosensors is given in Brian R. Eggins's chemical and biosensor, John Wiley & Sons, 2002, which is incorporated by reference in this application. The sensor 66 has various dimensions and shapes. Sensors with dimensions less than 3 microns are suitable for certain applications. It will be apparent to those skilled in the art that sensor 66 includes sensors that detect molecular concentration, temperature, osmotic pressure, pH, shear force, and the like. Such sensors are prior art and can be easily obtained or manufactured.

  The device 40 may be manufactured by interposing a substrate plate between the base plate 42 and the cover plate 44. Embedded in the substrate is a microfluidic network having fluid grooves and valves. Device 40 also has a transparent indium tin oxide conductor connected to the vertical connection wiring, which traverses the microfluidic network on the substrate. The network can be made by various methods known to those skilled in the art, for example, B.-H. Jo et al., “In Polydimethyl-siloxane (PDMS) Elastomers, incorporated herein by reference. Three-dimensional microgrooving ", Journal of Microelectromechanical Systems, Vol. 9, p76-81, 2000; J. Narasimhan, I. Papautsky," High-speed machining of high-temperature embossing tools using PDMS " , Micorfludics, BioMEMS and Medical Microsystems, International Society for Optical Engineering 4982 Proceedings, p110-119, 2003; M. Rudman et al., “Formation of near-field subwavelength fine patterns, pipettes Induced argon fluoride excimer laser microfabrication ", Journal of Apply Physics, 72, p4379-4383, 1992; B. Nabeth et al., "Dynamic mechanical properties of UV curable polyurethane acrylates with various reactive excipients", Journal of Applied Polymer Science, 60, p2113. -2123, 1996;

  The network may be configured by PDMS microstamping, hot embossing, silicon micromachining, laser ablation, or UV curing of materials such as isobornyl acrylate (IBA). PDMS is a suitable material and can be used to form molds used to mold PDMS layers using standard microfabrication techniques, with various grooves surrounding the chamber chamber 48 on the surface of the PDMS layer. It is stamped. The PDMS layers are stacked one above the other in a manner known to those skilled in the art to form sidewalls 45. Another layer of PDMS is configured such that one of the two components mixed to form PDMS is in excess. After curing, adjacent layers are melted by heat, and excess components in one layer react with excess components in the layers sandwiching this layer.

  The material of each sidewall 46 (i.e. its properties) is selected individually to produce the desired specific effect or to avoid undesirable specific effects. For example, to simulate the in vivo niche of stem cells, spermatogonia and Sertoli cells that support them are coated with collagen-IV and / or laminin-1. Laminin-1 is preferred because laminin is the dominant component of the basement membrane that partitions spermatogonia within the biological niche. Male germ stem cells are not tightly bound to collagen-IV, and the walls made of collagen-IV gel make it easier to control the orientation of cells growing in chamber chamber 48, so collagen-IV gel Is preferable. Collagen-IV gels are usually coated with binding proteins found in cell membranes and Sertoli cells are exposed to biological niches such as claudin, occludin, cadherin, connexin and selectin. The remaining inner surface of the chamber 48 is composed of a biological niche such as glass or polydimethylsiloxane (PDMS).

  Sidewall 46, particularly its contoured inner surface, is shaped using standard micromachining methods known to those skilled in the art and is incorporated, for example, as a reference in this application, "Micrographic Transfer, Micromachine Machining and Micromachining Handbook, ”Volumes 1 and 2, International Society for optical Engineering, P. Rai-Choudhury (Editor), 1997.

  The instrument 40 may be constructed of a transparent material that allows light to pass into the chamber 48 so that the cells are optically monitored, or the light is used to perform certain photochemistry within the chamber 48. The reaction is started.

  The instrument 40 may have other physical, chemical or electrical components necessary or preferred for a given purpose of cell culture. As will be apparent to the bioprocess engineer, for example, the device is connected to other devices and equipment for the proper operation of the culture process, which is described in “Biochemical Engineering”, Marcel Dekker Press, Blanche. (H. Blanche), D. Clarke, 1997.

  A control system (not shown) such as a computer or other automated device is used to monitor and control the operation of the instrument 40 and the resulting data is analyzed. The culture environment in the chamber 48 is dynamically adjusted based on collected information in real time. Media flow and metabolite concentrations are monitored and controlled. For example, the sensor 66 is connected to the control system using a transparent electrode and is used for simultaneous measurement of oxygen concentration and pH in the chamber 48. A plurality of sensors 66 may also be used to measure the gradient of a given substance within the chamber chamber. The feedback information includes the pH in the chamber 48, glucose and oxygen concentrations, temperature, osmotic pressure, and shear force.

  A microfluidic pump (not shown) may be connected to the groove to supply fluid to the chamber chamber 48 and drain it from the chamber chamber 48. Any suitable pump may be used. For example, the pump is incorporated by reference in the present application, A. Hatch et al., “Ferrofluid Magnetic Small Pump”, Journal of Microelectromechanical Systems, Vol. 10, p215-221, 2001; Soelensen (O Soerensen et al., "Micromachined Flux Handling Parts-Small Pumps", Proceedings, SPIE-International Society for Optical Engineering 3857, p52-60, 1999; P. Woias , "Small Pumps-Summary of the First 20 Years", Proceedings, SPIE-International Society for Optical Engineering 4560, p39-52, 2001; Examples of suitable pumps include ferrofluid slugs driven by rotating magnets around the ring chamber, piezoelectric crystal doors that seal the side pillars, piezoelectric crystal pumps, and the like. The flow rate of the electrokinetic pump in a groove with a cross-sectional area of 0 to 100 microns reaches 10 or 100 microns / second. The pumps are installed at each position of the network of grooves connected to the chamber 48, and flow resistance is suppressed particularly in long grooves.

  Prior to installing the cells, the surface of the chamber 48 may be given a positive or negative charge to control cell adsorption or behavior. The charge is applied by various means known to those skilled in the art, for example by exposure to oxygen gas at 400 ° C., exposure to acids or bases, or by rubbing with materials of different electron affinity.

  The inner surface of the chamber 40 may be reactive such that other molecules are covalently bound to it. Reactivity is imparted to the surface by various methods known to those skilled in the art. The reactivity is imparted by treatment with a molecule having an amine group on the surface, such as aminopropyltrimethoxysilane (APTS). Is done. A thin layer or bulk material may be coupled to the surface. Bulk materials include gels made of protein, polyacrylamide, or other substances. Such a gel may be formed in a mold using standard microfabrication techniques. The gel is placed on the sidewall and covalently bonded by reaction with the active surface. For example, collagen-IV gel or fibronectin is used. Since male germ stem cells do not bind tightly to fibronectin such as collagen-IV or laminin-1, some surfaces of chamber chamber 48 are coated with collagen-IV gel or fibronectin and other surfaces When coated with laminin-1, the cells in the chamber can be oriented as desired.

  The surface may be coated with binding protein, in which case the cell types of interest are usually claudins and occludins (for tight junctions), cadherins (for actin-binding, adsorptive binding), connexins (gap junctions) And in the natural environment such as selectins (in the case of selectin-lectin interactions). A thin layer of protein may be patterned on the inner surface of the chamber 40, e.g. by treatment of an APTS-treated surface with glutaraldehyde or photoactivated cross-linked 4-benzoylbenzoic acid succinimidyl ester. Or known to those skilled in the art, as Ito and Yoshihiro, “Surface micropatterning for cell function regulation”, Biomaterials, Vol. 20, p2333-2342, 1999, which is incorporated by reference in this application. Patterned using other techniques. The protein may be of any type. For example, when sperm cells are cultured, laminin-1 is used because it is the dominant component of the basement membrane in the in vivo niche of sperm cells. The protein may be patterned within a concentration gradient at the surface of the niche compartment in a manner known to those skilled in the art.

  The device 40 may be embedded with a conductor (not shown) to connect the sensor and pump electrodes to an external electronic device and a power source. The conductor is installed using standard microelectronic processing techniques. For example, the conductor is on the order of nanometer thickness. The conductor is placed along the surface of the substrate or through the substrate. The conductor may also be coated with an inert coating of a non-conductive material such as aluminum oxide.

  The niche chamber 48 is useful when culturing specific cell types, such as cells found in mammalian testis. As described above, chamber chamber 48 is used for culturing cell aggregates, where each cell has its own individually controlled niche, and the entire aggregate produces the desired biological effect and sperm Etc. are generated.

An example of how to configure the device 40 is as follows:
Preparing a substantially flat substrate made of silicon or silicon dioxide and forming the base plate 42;
Incorporating the sensor 66 into the base plate 42;
Installing a transparent conductor (not shown) of indium tin oxide or titanium nitride to connect the sensor 66 to an electrode on the outer surface of the base plate 42;
Forming an insulating layer (not shown) on top of the conductor, the insulating layer comprising, for example, silicon nitride, polyimide, or polysiloxane;
Forming and laminating several substrate layers to form sidewalls 46 having a microfluidic network and incorporating grooves on the surface of each layer as described above;
Covering the side wall 46 with a substantially flat substrate forming the cover plate 44, whereby the chamber chamber 40 is formed;
Before or after coating chamber chamber 40, optionally performed, with some internal surfaces of chamber chamber 40 being reactive and certain molecules being covalently bound thereto;
Patterning a thin layer of protein on several inner surfaces of chamber chamber 40, as needed, before or after coating chamber chamber 40;

  During operation, the chamber chamber 48 is populated with many desired seed cells. The base and cover plates 42, 44 constrain the cells between them and limit the movement of the cells towards or away from the base and cover plates.

  As shown in FIG. 8, in order to further restrain the target cell 68 or provide the desired microenvironment to the target cell 68, one or more other cells (eg, cells 70, 72 and 74) are placed in the chamber. It is installed around the target cell 68 in the chamber 48. In such a case, other cells help restrain the target cell. The target cell and the adjacent cell may be manually placed at a specific position in the chamber chamber 48. Or these may be installed by self-organization. It is clear that two adjacent cells are constrained to each other when they are compressed together. Therefore, the target cell also helps restrain neighboring cells.

  Cells are planted in the chamber chamber 48 by removing the cover plate 44 from the top and placing it using a micropipette. The cover plate 44 is set down slowly so as not to disturb the cells. The cover plate 44 is installed using standard alignment means. Cells may also be implanted through one or more fluid grooves 50-62. For example, the cells are supplied to the chamber 48 by one groove 50. Carrier fluid is removed through groove 62 by opening valve 64 in grooves 50 and 62. Once supplied, the cells are self-assembled or self-assembled in the chamber 48 so that the barrier in the chamber 48 has suitable preselected characteristics, such as appropriate affinity for a particular cell. Depending on the surface, these operations can be easily performed.

  After the seed cell is installed, the valve 64 in the groove 50 is closed.

  Chamber chamber 48 contains a fluid culture medium that passes through groove 52. For example, when culturing spermatogonia, suitable media include vas deferens replacement. The medium may contain high concentrations of amino acids, potassium, androgens and estrogens. It is clear that these media mimic the vas deferens necessary for the proper development of sperm flagella.

  The carrier fluid may be discharged from the groove 62.

  The supplemental culture medium is supplied to the chamber 48 through grooves 58 and 60 that function as source and sink grooves, respectively. For example, the supplemental medium includes testosterone, which is normally contained in the organism on the side of the basement membrane that faces the vas deferens lumen.

  The media flux and concentration are monitored and controlled. For example, using the sensor 66, the oxygen concentration and pH in the chamber 48 are measured simultaneously. It is also possible to measure the gradient of a given substance in the chamber 48 using a plurality of sensors 66.

  If the instrument 40 is transparent, cell growth in the chamber 48 can be monitored using, for example, a microscope imaging device. For example, such a device may be used to detect the presence of mature sperm, ie, sperm that is removed and sperm that is used for the intended purpose.

  A computer or other automated device may be used to monitor and control the operation of the instrument 40 and analyze the data obtained. The culture environment of the chamber 48 may be dynamically adjusted based on information collected in real time. The feedback information includes the pH, glucose and oxygen concentration in the chamber 48.

  FIG. 8 shows a specific example of cells cultured in the device 40. As shown, chamber chamber 48 has type A thin spermatogonia 68, type B thin spermatogonia 70, and two Sertoli cells 72. Although not shown in the figure, it is clear that each cell is constrained between them by a base and cover plates 42,44. As will be apparent to those skilled in the art, the niche of each cell growing in chamber 48 is close to the biological niche of such cells. Each cell is constrained by one or more other cells, and in some cases by one or more sidewalls 46. For example, cell 68 is constrained by cells 70 and 72 in the horizontal direction and by wall 46 and cell 74 in the vertical direction. In other words, cells 70 and 72 constrain cell 68 between them, which contact cell 68 on the opposite side and prevent cell 68 from moving away from these cells; 74 and bottom side wall 46 constrain cell 68 between them and contact cell 68 on the opposite side, preventing cell 68 from moving away from both.

  Cells in chamber 48 receive nutrients by diffusion through the space between the cells and, to a lesser extent, through the cells. Nutrients are also supplied through a groove placed at the top or bottom of the cell.

  If necessary, the cells may be removed from the culture chamber. To remove cells such as sperm from the chamber 48, the groove 50 is temporarily opened and the cells are carried out of the chamber 48 by the fluid carrier.

  One wall of the instrument 40 may have a septum, which allows access to the chamber chamber 48 using tweezers or a micropipette.

  The device 40 can be used for various purposes.

  For example, the device 40 can be used to generate healthy sperm with a male gene profile of interest in the unlikely event, for example, in the case of testicular cancer or infertility. Spermatogonia are obtained from a healthy subject and then stored in a controlled growth state as shown herein.

  In organisms, multi-cell units often have multiple cell types that exhibit specific functions, and these units often form physically distinct niche assemblies. Obviously, the device 40 can be used to provide and control an artificial niche assembly.

  The device 40 may also be used to provide a single niche for a single cell or a partial niche of a multi-cell unit. For example, when culturing the aforementioned sperm cells, instead of implanting Sertoli cells, Sertoli cells are used to construct the important proteins produced by Sertoli cells, such as Sry, inhibin and androgen binding protein, and the blood testis diaphragm. The device 40 may be configured and controlled to provide a boundary wall similar to that formed by.

  Obviously, the array of devices 40 may be fabricated on a single device. As shown, the chamber 48 may contain a plurality of cells or may be designed to culture a single target cell.

Another specific example is shown below.
(I) Automatic monitoring and control As described above, cell culture and monitoring may be controlled using a computer and a computer program, and the obtained data may be analyzed. A microprocessor may be attached to the incubator or another central unit. The computer system can record the measurement results from the sensors, analyze the data, and control the niche accordingly. Niches and accessory devices may be monitored and controlled by one or more processors and software programs.

  FIG. 9 shows an arrangement of data paths between the computer system 90 that controls the culture process and the culture control analysis system 92 as an example.

The niche environment is housed in a portable cartridge that has openings, electrical connections, mechanical connectors, and other features on the outer surface that are coupled to other devices. For example, the cartridge can be plugged into a device that performs a steady culture function, and this culture function can be concentrated at a certain distance from the immediate vicinity of each niche assembly. Such functions include storage of nutrients and gases, supply of positive or negative pressure to move liquids, solids or gases, reception and analysis of signals from sensors in the niche. The device has a multi-well plate and a cartridge is placed in each well. Alternatively, the cartridge is placed in a biosensor device that, for example, emits light through a niche and determines whether a cell represents a particular reporter gene. In yet another method, the cartridge is placed on a microscope stage or placed on a multiwell plate.
(Ii) Cell-scale molecular biological treatment within a single control niche Molecular biological means may be applied to cells within an artificial niche. This has several advantages. The cell is in a known state and in a defined environment. A cell culture device provides a “surgical room” for cells by providing life support to the cells, which allows the cells to withstand traumatic treatment, hold the cells in place, microinjection or electroperforation It is possible to precisely control the processing such as law. The culture apparatus may have a single niche or multiple niches.

  Biological, chemical analysis or other manipulation of single cell molecules is performed on individual cells in a defined and appropriate microenvironment using an exemplary cell culture instrument or apparatus of the present invention.

  10 and 11 show a culture apparatus 100 for shaping a niche chamber. The niche chamber has a pre-defined shape and dimensions and accommodates only one to three niches in at least one dimension.

  The niche chamber chamber has an inner surface 101, and a plurality of microfluidic grooves 103 adjusted by a valve 102 are connected to the niche chamber chamber. Another groove 103 may be used to supply or drain different solutions or substances from the chamber chamber, and the other groove 103 may have different dimensions and shapes. Groove 103 may be interconnected to adjacent cell culture units via groove 104. Grooves 103 and 104 introduce the vector, immune body, calcium chloride, lipofectin or some other ligand and distribute the medium to wash the cells. The auxiliary niche environment consists of anisotropy to the surface that remains in contact with the cells when handled, a biomimetic external cell matrix coating, suitable source and sink solutions, and the size and shape of the niche chamber chamber .

  Cells are placed or removed through the microfluidic groove 113 having valves 112, 124 while the solution is allowed to flow through the grooves 114 and 104. The valve 124 has a slider 122 that is slidably mounted between plates 105 and 109. Each of the plates 105 and 109 has a through hole (not shown) to allow access to the niche chamber and / or fluid communication with the niche chamber. The slider 122 can slide so as to enter and exit between the plates 105 and 109, and opens and closes the through hole. The slider 122 is operated by a floating bag 110, and this floating bag is controlled by a valve 111. The gel or filter 107 allows diffusion of the solution that simulates natural soaking of cells. Cells can also be introduced from the top or bottom of the niche chamber by installing with a micropipette or via a groove passing through, for example, plates 105 and 109.

  In the electroporation method, the electrode 108 forms an electric field across the cell. The electrode 108 is placed on the opposite side of the cell and the cells in the niche are electroporated. In the normal electroporation method, the cell membrane is damaged, so that cells are killed at a high level. In an artificial niche controlled in the manner described above, all cells can be supported on that side. By using this method, cells can be grown at a controlled copy number. A single vector is placed in close proximity to a single cell because it differs from the method that relies on the opportunity for these two to be linked together in a bulk solution.

  A piston 120 is provided and installed, which slides within the chamber chamber, thereby changing the volume of the chamber chamber, for example under the pressure of the bladder 117. Expansion and contraction of the float bag 117 is controlled by the groove 115 and the valve 106.

  Obviously, the volume of the niche chamber of the device 100 may be varied by other actuation devices than the inflatable bladder. For example, the piston 120 is actuated by other known actuation mechanisms. For example, the piston may be installed on a plate movable up and down by macroscopic means such as a screw, a lever, a clamp, a micrometer, and a piezoelectric crystal.

  For example, the inflatable bladder 117 compresses and ruptures the cells in the chamber chamber with the piston 120, and in some cases, for example, the contents of the cells are sampled using the grooves 114 at other locations in the same monolithic device 100. This is analyzed by microcapillary electrophoresis, RT-PCR, or other methods. Cultured cells are compressed or exposed to other treatments that rupture the cells, and the contents of the cells go through microfluidic grooves, microcapillary electrophoresis devices, single cell mRNA extraction devices, HPLC-mass spectrometry devices , And other analysis devices such as analysis means.

  Alternatively, the cultured cells may be compressed together and fused. Cell fusion is preferred, for example, when producing hybridomas. The inflatable bladder 117 compresses the two cells together and compresses the movable niche partition with the piston 120 to reduce the allowable volume. A fusion chemical such as polyethylene glycol or a cell rupture chemical such as a high salt concentration solution may be added via the groove.

  The culture device 100 may be integrally formed with other devices in a single device as in other embodiments of the invention. For example, as shown in FIGS. 12A and 12B, an assembly 126 of the device 100 is provided. In another embodiment, the assembly includes one or more other embodiments of the present application. Unit devices, such as device 100, are stacked on top of each other with or without spacers and with or without gas and / or media layers. As illustrated, each unit device is substantially plate-shaped, and the units may be stacked in parallel. Individual culture units may be arranged in 2D and 3D arrays. Devices may be aligned using slots and grooves or other intersecting shapes of adjacent devices. Groove 104 and electrical connection 119 are connected to counterparts of adjacent devices.

  The opening of the microfluidic groove comes into contact with the cultured cell, and the ion channel function can be detected by the cell patch clamp method.

  Microfluidic grooves sample niche components and supply them to devices that perform such analysis, such as microcapillary electrophoresis devices, gas chromatographic devices, high pressure fluid chromatographic devices, mass spectrometers, or other measuring devices. .

The fluid in the fluid channel 104 connected to two or more niche chambers is sampled and analyzed by a high performance liquid chromatography (HPLC) mass spectrometer, microcapillary electrophoresis device, or other analytical means.
(Iii) 3D tissue The problem with existing artificial organs is that these microstructures are insufficient. For example, existing artificial organs do not have hair follicles and sweat glands, and thus are not comfortable and unnatural.

  FIG. 13 shows a procedure for assembling cells into a three-dimensional (3D) pattern as one embodiment of the present invention. A plurality of artificial niches 131 are formed in a two-dimensional (2D) layer 132. Some of such layers 132 may be formed and cultured. Each layer 132 has a different niche 131 for culturing different cell types and is arranged in a natural 2D pattern of uncultured cells. Cells cultured in different layers 132 may have different cell types or patterns and grow individually. Layers 132 are laminated together to form artificial skin 137, which has the same 3D pattern as found in natural skin.

  In one example, cells in several patterned layers 132 grow independently, and each layer 132 corresponds to a layer of cells of the target tissue type. The niche pattern for cells is configured to correspond to the pattern of cell types found in a single cell thickness or a few cell thickness of skin. Each layer of cells is cultured while constrained between the top barrier 133 and the bottom barrier 134. Top and bottom barriers 133 and 134 provide cells with niche components similar to those normally exposed to natural cells by the top and bottom cell layers of the barrier in natural tissue. The top and bottom barriers 133 and 134 may be interconnected horizontally within the layers to form a niche boundary.

  In order to form the artificial skin tissue 137, the basal layer 132A corresponding to the subcutaneous tissue layer is first selected. It is also possible to remove the barrier 133A and expose the cells to the niche 131. Obviously, if the top barrier 133A is removed, the niche 131 is exposed, but still the niche is fed from the bottom. Next, a layer 132B, for example corresponding to the dermis layer, is placed on top of the layer 132A. The bottom barrier 134B for the layer 132B is treated, for example, by the introduction of a simple external cell matrix (ECM) protease on one side of the cell so that the installed cells are minimized. After removal of the top barrier (not shown) covering layer 132B, layer 132B is released from bottom barrier 134B by compressing the underside of each cell through a microfluidic groove (not shown). The To add another layer 132, the bottom barrier 134B is removed. This process is repeated and additional layers are installed. The laminated layers 132 are adhered to each other by a biological adhesive as commonly used in surgery. This series of layer placement and adhesion steps is repeated until the laminated layer forms an artificial skin tissue 137 that resembles a 3D tissue body of natural skin tissue. The structure through which natural tissues such as blood vessels and hair follicles pass vertically grows gradually in another layer 132 until the layers are aligned and stacked and the cells in tissue 137 adhere to each other and form a structure. .

  Vascular epithelial cell islands are cultured to facilitate angiogenesis and integration with the main vascular system. Therefore, blood vessels are rapidly formed and supply to the tissue 137 begins. Blood vessels that cross adjacent layers are formed by the growth of a ring of endothelial tissue in alignment with a series of layers. The nerve is also patterned. Neurons placed around the end of the layer with axons whose body extends into the layer eliminates the need for nerve distribution after organ synthesis. Fine structures such as hair follicles that cannot be regenerated using current tissue engineering techniques may be regenerated by this method.

  A layer of biodegradable polymer such as polylactic acid or polyglycolic acid, or a layer of extracellular matrix material such as a collagen sheet with appropriate pores, can be used before surgical tying and laminating the layers. It is placed on top of the layer 132 with glue and glued.

Some niches are designed such that when the upper barrier 133 is removed, the cells are connected to adjacent niches by extracellular matrix material through a groove that is open at the top.
(Iv) In vitro fertilization Oocytes can be grown in an artificial niche optimized for in vitro fertilization. Insert the tip of the micropipette into the niche and microinject the egg. The niche may expand to accommodate egg division.
(V) Long-term storage of cells (cell bank)
Currently, the most common method of storing cells for later use is to freeze the cells. The problem with the freezing method is that the cells are exposed to a naturally damaging environment, which can cause undesirable effects such as increased mortality.

Cells cultured in individually controlled artificial niche can be maintained in a specific state for a long time. The metabolic action of the cells can be slowed, for example, by keeping them at a low temperature above the freezing point. The cells may be protected from cosmic rays by shielding. The surface defining the artificial niche is selected to keep cells (such as stem cells or progenitor cells) in a non-differentiated state. For example, differentiation is suppressed or controlled by preventing cells from adsorbing to the barrier.
(Vi) Production of cell-derived products In conventional bioreactors used for the production of recombinant proteins or other biomolecules, there are adverse conditions that are harmful to cells and reduce product yield. It will be apparent to those skilled in the art. Common adverse conditions include accumulation of ammonia, lactate or ethanol; oxygen deficiency or excess; lack of glutamine, glucose or growth factors; low or high pH or temperature; other factors;

  When adverse conditions are detected, the individually controlled artificial niche is immediately improved. Thus, application of embodiments of the present invention facilitates the production of healthy cells and improves production yield and / or quality.

  Also, in conventional cell culture, media and cells are often agitated to reduce the local concentration within the culture chamber. Agitation produces a level of shear that is detrimental to the cells. In a controlled artificial niche, the culture medium nourishes the cells, so that the concentration gradient is controlled without agitation of the medium or cells, avoiding the harmful shear forces caused by agitation.

  In addition, the basic conditions of the niche (that is, the conditions of the cells when there are no harmful conditions) can be controlled to obtain a higher production yield than conventional bulk culture.

  One or more niches of the bulk culture apparatus are individually controlled and monitored. Cells cultured in a controlled niche can always be observed at high magnification and / or with a biosensor. In such cases, these cells are used to monitor overall bulk culture conditions.

Furthermore, optimized cell-scale niches can be arranged in small particles for niche-dependent cell culture in a standard stirred vessel bioreactor.
(Vii) “Organism on the chip”
Examples of the present invention can be used to fabricate a mutual support cell type system. Assemblies of individually controlled artificial niche assemblies of different cell types are connected to sub-networks that function as organ functions, and these sub-networks are connected to a large network that functions as an organ system of an organism. For example, a single device may control all the niche of different cell types found in the kidney, the niche of different cell types found in the liver, and the body organs.

  Cellular niches from different organism tissues are chemically connected to each other via a circulatory network simulating the organism. Niche systems regenerate the functions and physiology of an organism.

As an example, device 140 is shown in FIGS. 14A and 14B. This device has a single network. Obviously, the network may have a different topology than that shown in FIGS. 14A and 14B. The device 140 has a pump 142, a valve 144 and a fluid communication channel 146 that allows chemical communication within the niche chamber 14. The natural organism's digestive and circulatory systems are regenerated in the device 140 and the cells cultured in the device 140 can be supplied with more complex culture materials such as food.
(Viii) Niche optimized wrapping
One or more population niche may be directly connected to a natural layer of cells or a layer of natural tissue. For example, a niche layer is placed adjacent to and in contact with the treatment area of the patient, and the niche layers are each niche until the patient's natural tissue forms the upper boundary of the niche within the layer. Formed in the cell culture device with the top of the cell open.

In certain instances, such as gingival gingival growth on the teeth, bare dentin or enamel is covered by a niche wrapping attachment where the cells in the niche are exposed towards the tooth, and both adhere to the existing At the end of the wrapping attachment that faces the gingiva, the cells are exposed to the cells in the gingiva exposed by removing the surface layer. Cells in the niche adsorb to the tooth and other cells, which supply nutrients, mechanical support, growth factor gradients, and other niche components to all sides defined by the niche chamber To do. Tubes and valves are connected to the niche chamber and cells can move from existing tissue toward the niche. A specific example of this is shown in FIG. 15, which shows a wrap attachment 150 designed to support tissue regeneration. The tooth 151 takes root in the gum 153. Gingiva 153 suppresses exposure of tooth material 152. The mounting portion 150 defines a number of niche chambers 154 that are open on one side. The opened side is stuck to the tooth 152, and the cells (not shown) in the niche chamber 154 are adsorbed to the tooth 152 over a long period of time. Nutrients are supplied to the cells through a groove 159 from a medium reservoir 155 that is pressurized by the microfluidic pump 156 using energy from a power source 157. The attachment may be made of a flexible material such as PDMS, which is adapted to the surface of the tooth 152, the gum 153 and the enamel 158.
(Ix) Variable shape The shape of the niche chamber, as well as the shape of the artificial niche, may be controlled in order to adapt to changes in the shape of the cells in culture, such as the growth stage of differentiation.

  16A and 16B show an example of the niche chamber 161. This niche chamber changes shape to accommodate neural progenitor growth axons such as differentiation. The niche chamber 161 is connected to a tube 162 that houses a plunger 163 having a lumen 164.

  In operation, the niche chamber 161 is sealed with a circular neuron precursor. Molecules that attract the axon growth cone are supplied to the chamber 161 via the lumen 164 of the plunger 163. As the axon of the neuron moves towards the plunger tip, this tip is drawn into the tube 162. The tube 162 may be metric lengths if desired. A niche of cells, such as Schwann cells, can be juxtaposed to the tube 162 to myelinate axons. When the axon grows, the upper part of the niche chamber 161 is removed and the axon is captured. Molecules released from patch clamps at the corners of the niche chamber mimic the presence of synapses that stimulate neurons.

  The artificial niche may also control radial expansion. This is possible, for example, by surrounding the niche with a gel or solid, which is compressed and ruptured to grow and divide the cells.

  For example, in a culture device designed to accommodate growing follicles that will become ova in the future, the size of the niche chamber must be increased from about 10 microns to over 100 microns to accommodate only growing follicles. If it contains granular cells that surround the follicle and also a liquid-filled sinus adjacent to the ovum before ovulation, it needs to be larger.

  As an example, a cell culture device having an inflatable niche chamber is shown in FIGS. 17A, 17B and 18. The cell culture device 170 has an enclosure for receiving the gel 172, which defines the niche chamber 172. Gel 171 has two hemispheres 171A and 171B that are compressed together along boundary line 177. Gel 171 is placed in device 170 by a plunger (not shown) through groove 178. Grooves 173 and 174 connected to grooves 175 and 176 of apparatus 170 and through which gel 171 passes provide a path for liquid supply and discharge to and from niche chamber chamber 172. Tabs 179 are provided in contact with gel 171 to maintain grooves 173-176 in proper alignment.

In operation, the primary follicle is placed in the niche chamber 172 with a single layer of granular cells by a micropipette and micro-installer external to the apparatus 170. Gel 171 is composed of biomolecules in a pattern that mimics stromal cells in which natural granular cells are exposed in the ovary. An additional groove (not shown) open to the outside of the gel can also complement this function by diffusing the solution throughout the gel. The two half gels 171A and 171B are mated together to form a gel 171 that surrounds the installed follicle, and the gel 171 is introduced into the device 170 via a groove 178 using a plunger. The culture fluid is supplied to the niche in a manner that simulates the polarization gradient of nutrients and growth factors supplied to natural growing follicles exposed in the ovary because it is in a position corresponding to the blood supply in the bone marrow Is done. As follicles and granular cells grow, they push gel 171 and expand niche chamber 180, as shown in FIG. This mimics the swelling that occurs in the natural ovary due to the cells that push out the stromal cells in the ovary. To create an expanded cell space, the gel 171 may be composed of a compressible material, which is compressed by pressure from the cells. Alternatively, the gel 171 may be composed of digestible or consumable materials that are partially digested or consumed. For example, alginate or collagen gels are digested by arginase or collagenase, respectively. Damaged products may be drained by perfusion.
(X) Research on systematic information science Research on systematic information science was carried out using the restraint cell culture apparatus shown in FIGS. 19A and 19B.

  The culture chamber chamber of this apparatus was constructed using a 22 × 50 mm cover slip (as the bottom plate) and a 1.3 × 8 × 1 mm microscope slide (as the top plate). The ends of the microscope slide were glued to a coverslip and these were secured together using a small clamp. The adhesive used is a medical type A silicone adhesive (Dow Corning®). The adhesive was allowed to cure for 72 hours before exposure to the cells. After the adhesive was dried, the clamp was removed and a glass tube was adhered around the chamber chamber to form a well as a media reservoir, as shown in FIG. 19A. In order to prevent contamination and to minimize light scattering during imaging, it is beneficial to use a lid that just fits the top of the coverslip.

  Cells are prepared and cultured as follows.

Adult male CD1 mice (25-30g, Charles River, Quebec, Canada) were killed by cervical dislocation, and V. Tropepe et al. Neural stem cells were isolated by the method shown in Growth Factors-Alpha Zero and Conversion of Old Mice, Journal of Neuroscience, Vol. 17, p7850-7859 (Tropepe 97), 1997. This document is incorporated as a reference document of the present application. Briefly, excised brain, which 1.3MgCl _2, 26NaHCO _3, 10D- glucose, 124NaCl, 5KCl, and 2CaCl including ₂ (all in mM) were placed in oxygenated artificial CNS fluid vessel . The lateral ventricular robe is dissected and placed in an enzyme solution at 37 ° C. (1.33 mg / ml trypsin (Sigma), 0.67 mg / ml hyaluronidase (Sigma), and 0.2 mg / ml kynurenic acid (Sigma)). Installed for a minute. Tissue was transported into serum-free medium containing 0.7 mg / ml trypsin inhibitor (Roche®), which was centrifuged at 1500 rpm for 5 minutes. Remove suspended matter, 20 ng / ml epidermal growth factor (EGF, mouse lower jaw, Sigma®), 10 ng / ml linear fibroblast growth factor (FGF2, human recombinant, sigma), 2 μg / ml Of heparin (Sigma) and replaced with complete medium (CM) consisting of serum-free medium. The pellets were powdered using a sterile fire-polished Pasteurella pipette and centrifuged again at 1500 rpm for 5 minutes. The pellet was then resuspended in CM and the cells were cultured at a clonal density of less than 20 cells / μl.

  After the generation of primary neurospheres, the culture is shown in V. Tropepe et al., “Neural stem cell proliferation in growing mouse limbs to EGF and FGF”, Developmental Biology, 208, p168-88 (Tropepe 99), 1999. As described, bulk was passed every 5 to 7 days. This document is incorporated as a reference document of the present application. Briefly, neurospheres are collected, centrifuged at 1500 rpm for 5 minutes, resuspended in CM, triturated into a single cell suspension using a hot abrasive pipette and placed in the CM. Cells are placed at constant clonal density in tissue culture flasks (Nunc®, Naperville, Ill.).

Polystyrene microspheres (Polyscience (registered trademark)) having an average diameter of 5.8 μm are washed three times with nanopure water (registered trademark) and air-dried. The microspheres are sterilized under a UV lamp and resuspended in CM. Neurospheres are sampled 10 μL at the tip of a silicon pipette and triturated 5 to 15 times until turbidized in a sterile cover slip glass. The cell suspension is then filtered using a 40 μm sterilized nylon sieve (Becton Dickinson®). The cell density of the filtrate is adjusted to 10 ⁶ cells / mL. Microspheres are added at a density of 3 × 10 ⁶ microspheres / mL. The upper plate is lifted with rubberized tweezers and a 2 μL cell / microsphere suspension is placed at the chamber chamber end, allowing the suspension to flow into the gap by capillary force. The upper plate is then slowly lowered over a period of several seconds. Excess cell suspension released when the top plate is lowered is pipetted from the opening in the gap between the top and bottom plates. The wells are then filled with 1 mL of fresh CM and the cells grow.

  After fixation, the stirrer is agitated at 120 rpm so that the reagent is transported to the constrained culture space in the culture chamber, and washing and culture are performed. Cells are washed and fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.3% Triton X-100 for 5 minutes, and fixed in 10% goat serum for 1 hour. The cells can be rabbit anti-GFAP (Chemicon®), mouse anti-β-tubulin III IgG (Sigma), mouse anti-O4 IgM (Chemicon®), mouse anti-nestin IgG, or mouse anti- Incubate with S100 IgG for 1 hour. After soaking the primary immunity, the cells were washed for 2 hours and then the appropriate secondary immunity: AMCA-conjugated goat anti-rabbit IgG, fluorescein-conjugated goat anti-mouse IgM, and londamine-conjugated goat. Incubated with anti-mouse IgG (Jackson Immunoresearch®) for 30 minutes. The cells were incubated with room temperature rhodamine-conjugated phalloidin (Molecular Probes®) for 30 minutes.

Cultured cells were imaged with an inversion microscope (Zeiss, Axiovert200®) using a DIC optical device. The cell culture chamber was maintained in a 5% CO ₂ humidified air environment at 37 ° C. Samples were irradiated every 3 minutes during image acquisition and were kept dark otherwise. Images were captured with a digital camera (Sony (registered trademark)). The cells were first imaged at low magnification (5 ×) until a single cell was confirmed to divide, and subsequent colony development was imaged at 40 × magnification.

  Systematic information science information was obtained as follows. A large number of image frames were acquired in a fixed period, for example, in 109 hours. The location of each cell within each frame of the image sequence was stored and changes in the microenvironment, behavior, and morphology of the experimental subject were recorded. Obtained values such as speed and proximity to other cells or colonies were calculated using a computer and specially developed software. The combined data sets were queried and analyzed in the phylogenetic tree using many 2D and 3D fields of view, pattern search, and cluster analysis.

  With DIC or confocal imaging methods, it was difficult to reliably identify individual cells within the neurosphere when the surface depth exceeded about 15 μm. To overcome this limitation, dissociated UNC was constrained between parallel glass plates separated by 5.8 μm polystyrene microspheres. Growing colonies are referred to as “neurodiscs” because the cells are geometrically constrained to a single layer within the chamber chamber to form a roughly circular colony. This constrained geometry culture system allows long-term, high-resolution viewing of all cells in the growing colony. The full development of clonal derived colonies is monitored with images taken every 3 minutes. FIG. 20 shows an image of a colony of 26 cells. This is the growth situation after 5 days from a single cell. During culture, it is observed that three cells have died and one cell has moved from the field of view. Individual cells do not move, rotate or disperse much when not touching another cell. On the other hand, cells in contact with other cells constantly move and expand each other. This means that the constraining surface does not support sufficient cell adsorption for migration or dispersion. Therefore, this culture system provides effective non-adhesion. Cell migration on the surface of the neurosphere is also observed using low speed imaging techniques.

  Since the growth of cells is restricted in a space of 5.8 μm thickness, details of the cell interface can be observed. In addition, vesicles and vesicle transport are observed, and cell tracking is extremely easy. Around some discs, cell bubbles (ie, the formation of small membrane bubbles along the cell boundaries) are sometimes observed.

  To follow the development of a single cell-derived disc, DIC images are collected over 109 hours. Images are analyzed to obtain lineage relationships of cultured cells. For example, cell location, morphology, and behavior are tracked within each series of images. A complete series of image records allows the reconstruction of the colony history. From the manual intrusion location and phenotypic data, derived characteristics including location within the cell population, closest history, velocity, and cell area are calculated.

  In constrained culture geometry, the spindle axis is always perpendicular to the observation axis, facilitating the study of division symmetry. Parameters that can be measured using this culture device include the relative size and shape of the daughter, the location of cell division relative to the population of cells, the orientation of the spindle axis, the chromosome orientation, and the distribution of components between the cytoplasm and sister. It is. Each of these variables is used in phylogenetic analysis of sister cell fate comparisons.

  It will be appreciated that embodiments of the present invention have several advantages over conventional culture devices and methods.

  For example, by placing a particular desired molecule or cell adjacent to a subject cell and / or by removing a particular neighboring cell from an artificial niche, for example, the artificial provided by the embodiments of the present invention Within a niche, single cells with precisely controlled properties can be cultured. It is also possible to control the concentration gradient of nutrients and oxygen gas in the culture medium. They are used to facilitate the orientation and placement of certain cells (eg, cells may be moved in a desired direction to implant source material). Thus, the niche provided by the device reflects a biological niche.

  In various embodiments, it is also possible to monitor niche and single cell level cell growth for a long period of time, for example, to acquire and record a complete growth history for a single cell. Thus, accurate knowledge about the biological properties of the growing cell, including the correct phenotype, can be obtained. Furthermore, by constraining the cells between the barriers, cell growth and small cell details of live cells can be observed directly, including standard bright field imaging devices or fluorescent imaging devices. Imaging techniques are used, and no confocal microscope or laser imaging device is used. Obviously, when the cells are constrained in the vicinity of the focal plane of the objective lens, the image quality is significantly improved compared to the image of the cells that have moved away from the focal plane. Experimental results show that images of cells cultured within a one-dimensional boundary on the order of 5 μm have a high resolution below the cell level. In addition, a high-resolution image can be obtained without exposing cultured cells to high-intensity light that is necessary when using conventional high-resolution imaging technology such as confocal imaging technology. As a result, these cells are restrained and imaged for days (such as one to several weeks) without any apparent adverse effect on the cells (decreased germination or altered material growth rate). It becomes. Therefore, restraint cells can be imaged for a long time with a high resolution technique such as differential interference contrast (DIC). Laser-operated confocal microscopy provides the same resolution as fluorescently labeled cells, but, as mentioned above, laser-induced phototoxicosis significantly shortens the imaging period of living cells imaged using this technique. To do. Accordingly, by imaging a sample of cultured cells using the example described above, the period can be prolonged compared to the case where the same sample is imaged using a confocal microscope. Also, the non-confocal imaging methods described above are less expensive than those normally used in other conventional high resolution methods such as confocal imaging methods or multi-photon imaging methods and Nipcor disc imaging methods. Can be implemented.

  When a collection of cells is constrained to a single layer, it is easy to remove specific cells or groups of cells from the collection. Therefore, the number of cultured cells obtained can be controlled, and for example, cells having a certain unfavorable phenotype can be prevented from being present in the obtained aggregate.

  Other features, advantages and significance of the present invention not previously shown will be apparent to those skilled in the art from the description and accompanying drawings.

  It will be apparent to those skilled in the art that many modifications can be made to the embodiments of the invention illustrated herein.

  For example, the spacer 34 may be changed to another spacing member. A non-spherical spacer may be used. The barrier plates may be separated by a grid-like flat spacer sandwiched between them, in which case a plurality of niche chambers are shaped.

  It is also possible to install an array of niche chambers 30 inside the chamber 26.

  The bottom barrier plate is not necessarily the same as the base plate 22 constituting the container 25. Alternatively, the bottom barrier plate and the base plate may be two separate plates so that the bottom barrier plate is removably installed in the container 25 and both plates are removable from the chamber 26.

  As described above, an additional barrier member, such as a weir, is detachably or fixedly installed on the container adjacent to the cover plate, and the media liquid flux is partially or completely shielded. The gradient of the medium in the culture chamber may be controlled, or a unidirectional gradient may be formed in the chamber.

  It is also possible to install an array of culture niche chambers 30 in one medium container 25.

  Although the configuration and operation of the device 40 has been illustrated with cultured spermatogonia as an example, if necessary, the device 40 may be modified to form a suitable or preferred microenvironment for different cell types. This will be apparent to those skilled in the art.

  Possible dimensions of the niche chamber 30 or 48 range from 100 nm to 10 cm.

  The culture chamber or niche chamber may be compressed for certain applications.

  During cell culture, the inner surface of the niche chamber 30 or 48 may be controlled dynamically, for example with a surface coating that becomes water repellent or hydrophilic upon application of a voltage potential. During cell growth or division, the surface shape may be changed, for example, using an inflatable float or moving the wall using an actuator such as a piezoelectric crystal.

  Other features that control or monitor the niche before or during culture include, but are not limited to, the freedom to diffuse molecules to or from the cells; the freedom to flow liquids; gases, liquid molecules, organisms Concentration of molecular or other macromolecules, signal generating molecules and other molecules; intensity and concentration gradients of these molecules; provision of such molecules that adsorb to surfaces, other molecules and other cells; pH; shape Hardness, selective permeability, and chemical modification of the vicinity of or in contact with the cell; the amount of dynamic movement of the cell neighbor; the density of cells surrounding the niche; the cell type in the vicinity of a given cell; included.

  Cells are placed in or removed from the culture chamber or niche chamber by centrifugation, movement of magnetic particles, or attraction by chemoattractants.

  A temporary partition plate may be installed to prevent cells from moving to an area of the culture space until the partition plate is moved or broken. For example, cell types can grow or expand until the divider is removed, or contact between two cells is avoided. The divider is removed or broken by hydraulic, mechanical, electrical, chemical or other means.

  Culture of solid materials, eg microspheres used in spacers with special coatings, or for other purposes, by microfluidic grooves, micropipettes, film formation, optical tweezers or other means Sometimes it may be introduced into the culture space.

  The culture chamber chamber or niche chamber chamber may be mutagenized, for example, by having a radiation source or by exposing them to short wavelength light. This is significant when conducting a forced evaluation experiment.

  Examples of the present invention are utilized to culture any type of cell, including mammalian cells such as prokaryotic and eukaryotic cells, stem cells, progenitor cells, terminal cells, terminally differentiated cells, transformed cells or embryonic cells. May be.

  Embodiments of the present invention may be used to verify whether a cultured cell is suitable for medical use (behaviorally, morphologically and environmentally) by logging the history of the cultured cell.

  It is also possible to provide electrodes to devices 10 and 40 to detect the electrical responsiveness of cells such as neurons that respond to the presence of molecules or other stimuli of interest. For example, an electrical or fluorescent response produced by cells within a niche may be used to detect neurotoxins or other bioactive agents.

  Embodiments of the present invention may be implanted in an organism. Use of such transplantation devices includes the secretion of beneficial biomolecules that respond to the major organ environment or the filtration of blood, lymph or other fluids.

  The cells may be cultured according to or using embodiments of the present invention and used for artificial tissue, organ, cell transplantation or in vitro fertilization.

  It is clear that the foregoing description is only an example to illustrate possible uses and variations of embodiments of the present invention. Accordingly, the present invention is defined by the following claims and is intended to embrace all such variations that are within the scope of the invention.

  This application claims priority from US Provisional Application No. 60 / 530,614, filed Dec. 19, 2003, entitled “Cell Culture Apparatus and Methods”.

1 is a perspective view of a cell culture device according to an embodiment of the present invention. FIG. 2 is a partial top view of the device of FIG. FIG. 3 is a front view of the portion shown in FIG. FIG. 2 is a partial front view of the device of FIG. 1 in operation. 2 is a differential interference contrast (DIC) image of cells cultured in the apparatus of FIG. FIG. 6 is a top sectional view of a cell culture device according to another embodiment of the present invention. FIG. 7 is a schematic front sectional view of the device of FIG. FIG. 7 is a schematic top view of the device shown in FIG. 6 during operation. It is a schematic block diagram which shows the path | route of the data between a control system and a cell culture analysis system as one Example of this invention. FIG. 6 is a schematic top view of a cell culture device according to another embodiment of the present invention. FIG. 11 is a schematic front view of the device of FIG. It is a schematic perspective view of the assembly of several cell culture equipment. FIG. 12B is a schematic top view of the assembly of FIG. 12A. It is the schematic which shows the structure of the cell aggregate | assembly by one Example of this invention. 1 is a schematic top view of a cell culture device according to one embodiment of the present invention. FIG. FIG. 14B is a schematic front view of the cell culture device of FIG. 14A. FIG. 3 is a schematic diagram showing application of a winding attachment according to an embodiment of the present invention. 1 is a schematic top view of a cell culture device according to one embodiment of the present invention. FIG. FIG. 16B is a schematic front view of the device of FIG. 16A. 1 is a schematic top view of a cell culture device according to one embodiment of the present invention. FIG. FIG. 17B is a schematic front view of the device of FIG. 17A. FIG. 17B is a schematic top view of the device of FIG. 17A in operation. It is a photograph of an actual cell culture device. It is a photograph of an actual cell culture device. FIG. 20 is a DIC image of cells cultured in the apparatus of FIG.

Claims (119)

Constraining a cell between a first and second barrier, wherein the barrier is spaced a distance equal to the size of the cell so as to contact the cell, wherein the cell is the first And a step that is prevented from moving toward or away from each of the second barriers; and
Placing one or more spacers between the first and second barriers to prevent the first and second barriers from over-compressing the cells;
Providing a culture material to the cells;
A method of cell culture, comprising: The first barrier is moved adjacent to the second barrier;
The restraining step and the placing step include at least partially separating the first and second barriers, the cell between the first and second barriers, and the one or more The method of claim 1, comprising introducing a spacer.   3. The method according to claim 1, wherein the one or more spacers are hard spheres, and each has a diameter equivalent to the dimension of the cells.   4. Each of the barriers has one or more features selected to mimic the characteristics of the biological niche of the cell. Method.   5. The method according to any one of claims 1 to 4, wherein the providing step comprises constructing a preselected concentration gradient of the culture material in the space.   6. The method according to any one of claims 1 to 5, further comprising the step of manually placing a specific cell type adjacent to the cell.   7. The method according to claim 1, wherein the first and second barriers include first and second plates.   The method according to any one of claims 1 to 7, wherein a monolayer of cells is cultured between the barriers.   9. The method of claim 8, further comprising the step of removing the cell from the monolayer of the cell when the cell is observed to meet one or more predetermined criteria. The method described.   10. The method of claim 9, wherein the one or more criteria are related to one or more of a karyotype, form and size. further,
11. The method according to claim 1, further comprising the step of limiting the number of cells surrounding the cell in order to enable control of the characteristics of the cell.   12. The method of claim 11, wherein the cells surrounding the cells are selected to provide a selected surface adjacent to the cells.   3. The method according to claim 2, wherein the introducing step comprises introducing the cell suspension and the one or more spacers between the first and second barriers. . Furthermore, with an elastic fixing agent, the step of fixing the end of the second barrier to the first barrier,
The method according to claim 2, wherein the first barrier is moved adjacent to the second barrier by the elastic fixing agent.   The cells are constrained between the surface of the first barrier and the surface of the second barrier, and each surface is selected to inhibit the cells from adsorbing to each surface; 15. A method according to any one of claims 1 to 14, characterized in that   The one or more spacers, together with the first and second barriers, are arranged to form a space having a shape simulating the shape of the biological niche of the cell. The method according to any one of 1 to 15.   17. The method according to claim 1, wherein the providing step includes the step of supplying the culture material to the cell at a predetermined rate.   8. The method of claim 7, wherein the plate is optically transparent.   The providing step comprises the step of permeating the culture material through one or both of the first and second barriers to between the first and second barriers. 19. A method according to any one of claims 1-18.   And detecting one or more of molecular concentration, temperature, osmotic pressure, pH and shear force using a sensor installed adjacent to the space between the first and second barriers. 20. The method according to any one of claims 1 to 19, comprising:   16. The method of claim 15, wherein the surface of the first barrier has a first type of molecule and the surface of the second barrier has a second type of molecule. . A method of manufacturing an artificial tissue,
Placing a monolayer of a first cell on a monolayer of a second cell;
Interacting the cells of the first monolayer with the cells of the second monolayer;
Have
22. The method according to claim 1, wherein each of the first and second monolayers is cultured according to the method according to any one of claims 1 to 21. A combination of a cell culture device and a cell culture,
First and second barriers and one or more spacers,
Between them, the desired cell culture space is shaped,
The barrier contacts a cell or cell body constrained in the culture space;
The spacer is sufficiently rigid and inhibits movement of the first and second barriers and is close to the dimensions of the cell or cell body;
The distance between the first and second barriers is equal to the size of the cell or cell body to be cultured;
A spacer that prevents the first and second barriers from over-compressing the cell or cell body; and first and second barriers and one or more spacers;
Means for providing culture material to the culture space at a predetermined rate;
A combination of a cell culture device and a cell culture.   24. The combination of claim 23, wherein the means for providing the culture material to the space has one or more fluid pathways through which fluid can flow into and out of the space. .   25. The combination of claim 24, wherein the one or more fluid paths have one or more microfluidic grooves, each groove terminating adjacent to the space.   26. A combination according to any one of claims 23 to 25, further comprising means for regulating fluid flow into or out of the space.   27. A combination according to any one of claims 23 to 26, wherein at least one of the at least two barriers is permeable to nutrients and gases.   28. The combination according to any one of claims 23 to 27, further comprising means for monitoring the cells constrained in the space.   29. A combination according to claim 28, wherein the means for monitoring comprises a sensor installed in the chamber chamber.   30. The combination according to claim 29, wherein the sensor is a sensor that detects one or more of molecular concentration, temperature, osmotic pressure, pH, and shear force.   31. A combination according to claim 29 or 30, further comprising one or more transparent electrodes connecting the sensor to a control system.   32. A combination according to any one of claims 23 to 31, wherein at least a part of one of the barriers is transparent.   33. A combination according to claim 32, wherein the barrier comprises a microscope cover slip.   The combination of claim 32, wherein the portion of the barrier is comprised of one of polystyrene, porous glass or other contact lens material.   35. A combination according to any one of claims 23 to 34, wherein at least one of the barriers is movable to adjust the size of the space.   36. The combination of claim 35, further comprising an actuator that moves the at least one of the barriers.   38. The combination of claim 36, wherein the actuating device comprises one or more of an inflatable bladder, a screw, a lever, a clamp, a micrometer, and a piezoelectric crystal.   38. The combination according to any one of claims 23 to 37, wherein the one or more spacers are removable from the first or second barrier.   38. The combination according to any one of claims 23 to 37, wherein the one or more spacers are attached to the first or second barrier.   38. The apparatus according to any one of claims 23 to 37, further comprising a partition plate that partitions the chamber chamber into a plurality of regions, wherein the cells are prevented from moving between the regions. combination.   41. The combination according to claim 40, wherein the partition plate is removable from the container.   The combination according to any one of claims 23 to 41, wherein the surface of the barrier has different molecular species.   24. A permeable membrane installed so as to cover an opening adjacent to the space, wherein the cells are prevented from being released from the space through the opening. 42. The combination according to any one of 42.   44. The combination according to any one of claims 23 to 43, wherein the barrier forms a plurality of spaces that restrain a plurality of cells.   45. The combination according to any one of claims 23 to 44, wherein the combination is contained in a cartridge.   46. The combination according to any one of claims 23 to 45, further comprising a fluid culture medium housed in the chamber chamber in which the cells are immersed.   48. The combination of claim 46, wherein at least one wall of the container has a septum that allows access to the space with a syringe or pipette.   48. The combination according to any one of claims 23 to 47, further comprising a capillary groove for transporting a cell-containing fluid into or out of the space. A combination of a cell culture device and a cell culture,
A container defining a chamber chamber containing a fluid culture medium;
At least two barriers defining a space in the chamber chamber, each of the barriers having at least two partition walls having one or more preselected characteristics;
An assembly of two or more cells constrained in the space, the assembly of two or more cells held in the space in continuous contact with each of the barriers during culture; and
Means for providing culture material to the space at a predetermined rate;
A combination of a cell culture device and a cell culture.   50. The combination of claim 49, wherein the space is sufficiently small to control the characteristics of individual cells in culture.   51. The combination according to claim 49 or 50, wherein the aggregate has a monolayer of cells.   52. The means for providing a culture material to the space has one or more fluid pathways through which fluid can flow into and out of the space. Combination described in one.   53. The combination of claim 52, wherein the one or more fluid paths have one or more microfluidic grooves that terminate adjacent to the space.   54. A combination according to any one of claims 49 to 53, further comprising means for regulating fluid flow into or out of the space.   55. A combination according to any one of claims 49 to 54, wherein at least one of the at least two barriers is permeable to nutrients and gases.   56. The combination according to any one of claims 49 to 55, further comprising means for monitoring the cells constrained in the space.   57. The combination according to claim 56, wherein the means for monitoring comprises a sensor installed in the chamber chamber.   58. The combination according to claim 57, wherein the sensor is a sensor that detects one or more of molecular concentration, temperature, osmotic pressure, pH, and shear force.   59. A combination according to claim 57 or 58, further comprising one or more transparent electrodes connecting the sensor to a control system.   60. A combination according to any one of claims 49 to 59, wherein at least part of one of the barriers is transparent.   61. The combination of claim 60, wherein one of the barriers is a microscope cover slip.   62. The combination of claim 61, wherein the portion of the barrier is comprised of one of polystyrene, porous glass, or other contact lens material.   63. A combination according to any one of claims 49 to 62, wherein at least one of the barriers is movable to adjust the size of the space.   64. The combination of claim 63, further comprising an actuator that moves the at least one of the barriers.   65. The combination according to claim 64, wherein the actuating device comprises one or more of an inflatable bladder, a screw, a lever, a clamp, a micrometer, and a piezoelectric crystal.   66. The method according to claim 49, further comprising one or more spacers disposed between the barriers, wherein the barriers prevent the cells from being excessively compressed. Combination described in one.   68. The combination of claim 66, wherein the one or more spacers are attached to one or more of the barriers.   68. The apparatus according to claim 49, further comprising a partition plate that divides the chamber chamber into a plurality of regions, wherein the cells are prevented from moving between the regions. combination.   69. The combination according to claim 68, wherein the partition plate is removable from the container.   70. The combination according to any one of claims 49 to 69, wherein the surface of the barrier has different molecular species.   49. A permeable membrane installed so as to cover an opening adjacent to the space, wherein cells are prevented from being released from the space through the opening. The combination according to any one of 70.   72. The combination according to any one of claims 49 to 71, wherein the barrier forms a plurality of spaces constraining a collection of a plurality of cells.   73. The combination according to any one of claims 49 to 72, wherein the combination is housed in a cartridge.   74. The combination according to any one of claims 49 to 73, further comprising a fluid culture medium accommodated in the chamber chamber, in which the cell aggregate is immersed.   75. A combination according to claim 74, wherein at least one wall of the container has a septum that allows access to the space with a syringe or pipette.   76. The combination according to any one of claims 49 to 75, further comprising a capillary groove for transporting fluid into or out of the space.   An aggregate of cells comprising cells cultured according to the method of any one of claims 1 to 21.   A cell cultured according to the method of any one of claims 1 to 21.   78. The cell aggregate according to claim 77 or the cell according to claim 78, which is used for artificial tissue, organ, cell transplantation or in vitro fertilization. A device for culturing cells in a controlled environment,
i) First and second barriers and one or more spacers, the desired constraining space being shaped between them, the distance between the barriers being equal to the size of the cell or cell body to be cultured The barrier is in contact with the cell or the cell body and prevents their movement, and the spacer is sufficiently rigid to suppress the movement of the first and second barriers, and the first and second barriers A first and second barrier and one or more spacers, wherein the cell or cell body is prevented from over-compressing;
ii) the inner surface of one or both of the first and second barriers having one or more properties selected to mimic the characteristics of the biological environment of the cells;
iii) means for providing culture material to the space;
Having equipment.   81. The device of claim 80, wherein the barrier has two opposing glass plates.   82. A device according to claim 80 or 81, wherein the space has dimensions comparable to the dimensions of a single cell.   83. The device according to any one of claims 80 to 82, wherein the space restricts cultured cells to a monolayer.   84. Apparatus according to any one of claims 80 to 83, wherein the means for providing comprises one or more fluid pathways through which fluid can flow into and out of the space. .   85. The apparatus of claim 84, wherein the one or more fluid paths have one or more microfluidic grooves that terminate adjacent to the space.   86. Apparatus according to any one of claims 80 to 85, further comprising means for regulating the flow into or out of the space.   87. A device according to any one of claims 80 to 86, wherein at least one of the barriers is permeable to nutrients and gases.   The device according to any one of claims 80 to 87, further comprising means for monitoring cells constrained in the space.   90. The apparatus of claim 88, wherein the means for monitoring includes a sensor installed in the chamber chamber.   90. The apparatus of claim 89, wherein the sensor is a sensor that detects one or more of molecular concentration, temperature, osmotic pressure, pH, and shear force.   The apparatus of claim 89 or 90, further comprising one or more transparent electrodes connecting the sensor to a control system.   92. A device according to any one of claims 89 to 91, wherein at least a part of one of the barriers is transparent.   93. The instrument of claim 92, wherein one of the barriers is a microscope cover slip.   94. The device of claim 93, wherein the portion of the barrier is comprised of one of polystyrene, porous glass, or other contact lens material.   95. A device according to any one of claims 80 to 94, wherein at least one of the barriers is movable to adjust the size of the space.   96. The device of claim 95, further comprising an actuator that moves the at least one of the barriers.   99. The apparatus of claim 96, wherein the actuating device comprises one or more of an inflatable bladder, a screw, a lever, a clamp, a micrometer, and a piezoelectric crystal.   98. The device according to any one of claims 80 to 97, wherein the one or more spacers are removable from the first or second barrier.   98. The device according to any one of claims 80 to 97, wherein the one or more spacers are attached to the first or second barrier.   99. The apparatus according to any one of claims 80 to 98, further comprising a partition plate that partitions the chamber chamber into a plurality of regions, wherein the cells are prevented from moving between the regions. machine.   100. The device of claim 99, wherein the partition plate is removable from the container.   102. A device according to any one of claims 80 to 101, wherein the surface of the barrier has different molecular species.   103. A permeable membrane installed so as to cover an opening adjacent to the space, wherein cells are prevented from being released from the space through the opening. The apparatus as described in any one of.   104. The device according to any one of claims 80 to 103, wherein the barrier forms a plurality of spaces that restrain a plurality of cells.   105. The device according to any one of claims 80 to 104, wherein the device is housed in a cartridge.   106. The apparatus according to any one of claims 80 to 105, further comprising a fluid culture medium accommodated in the chamber chamber.   107. The device of claim 106, wherein at least one wall of the container has a septum that allows access to the space with a syringe or pipette.   108. Apparatus according to any one of claims 80 to 107, further comprising a capillary groove for transporting fluid into or out of the space. A cell culture assembly having a plurality of devices, comprising:
79. An assembly wherein each of the devices is one of a combination according to any of claims 23 to 79 and a device according to any of claims 80 to 108.   110. The assembly according to claim 109, wherein each of the plurality of devices has a substantially plate shape, and the plurality of devices are stacked in parallel.   Culturing one or more cells while inhibiting the movement of the one or more cells, each of the one or more cells being continuously with two opposing barrier surfaces A method of cell culture comprising the steps of contacting and moving between said barrier surfaces.   112. The method of claim 111, wherein the barrier surfaces are substantially parallel to each other.   113. A method according to claim 111 or 112, wherein the barrier surface is substantially flat.   114. The method according to any one of claims 111 to 113, further comprising acquiring one or more images of the one or more cells during the culture.   115. The method of claim 114, wherein the one or more images are acquired using a non-confocal imaging device.   115. The method of claim 114, wherein the one or more images are acquired using a bright field imaging device or a fluorescence imaging device.   117. The method of claim 116, wherein the one or more images are acquired using a differential interference contrast (DIC) imaging device.   118. A method as claimed in any one of claims 114 to 117, wherein the one or more images comprise a plurality of images acquired over a period longer than about one day.   The one or more images are acquired over a long period of time without being noticeably or substantially limited by phototoxic effects that interfere with collecting information about the history of the behavior of one or more cells. 118. A method as claimed in any one of claims 114 to 117, comprising a plurality of images.

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